Variable expression of cyanide detoxification and tolerance genes in cyanogenic and acyanogenic white clover (Trifolium repens L.)

Premise of the study: β-cyanoalanine synthase (β-CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN) stress.  These functions are critical for all plants, as HCN is produced at low levels during basic metabolic processes, but are likely to be especially important in cyanogenic species, which release high levels of HCN following tissue damage. However, their expression has not been examined in cyanogenic species, nor has it been compared between cyanogenic and acyanogenic genotypes within a species.
Methods: We used a natural polymorphism for cyanogenesis in white clover to examine β-CAS and Aox gene expression in relation to cyanogenesis-associated HCN exposure.  We identified all β-CAS and Aox gene copies present in the genome, including members of the Aox1, Aox2a and Aox2d subfamilies previously reported in legumes.  Expression levels were compared between cyanogenic and acyanogenic genotypes, and under condi..., All qPCR protocols followed MIQE guidelines, including for numbers of biological and technical replicates and reaction optimization protocols.  Gene expression level was quantified using the CFX96 Real-Time PCR Detection System (BIO-RAD, Hercules, California, USA). For all genes, reactions included 1× iTaq™ Universal SYBR® Green Supermix, 0.5 µM (0.3 µM for β-CAS) forward primer, 0.5 µM (0.3 µM for β-CAS) reverse primer and 4-10 ng cDNA in a 20 µL reaction volume. Ef1α was used as the reference gene. PCR conditions followed the standard SYBR® Green cycle in CFX96 Real-Time PCR Detection System with slight modifications to maximize reaction efficiency: 95°C for 30 s, then [95°C for 5 s, 60°C (63°C for β-CAS) for 30 s] × 40 cycles. Three technical replicates were applied to each cDNA of the sample. The data were analyzed by the ΔΔCT method with efficiency calibration in CFX Maestro™ Software (BIO-RAD, Hercules, California, USA) according to the manufacturer’s instructions.,